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One-step 3'-End RNA Labeling with AF (Alexa Fluor®)-Dyes

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One-step labeling of short 3'-End AF (Alexa Fluor®)-Dye modified RNA is now easily achievable via T4 RNA Ligase 1-mediated incorporation of correspondingly labeled pCps (Fig. 1).

AF (Alexa Fluor®)-Dyes are hydrophilic fluorescent dyes with remarkable photostability. Linker-optimized AF (Alexa Fluor®)-Dye labeled pCps possess excellent substrate properties and thus allow efficient preparation of AF (Alexa Fluor®)-Dye modified RNA probes (up to 100 nt) (Fig. 1) (Tab. 1).

Figure 1: 3’-End AF(Alexa Fluor®) RNA labeling can be performed in a one-step procedure.

Figure 1: 3'-End AF (Alexa Fluor®) RNA labeling can be performed in a one-step procedure.
RNA oligo (43 nt) was 3'-End labeled using T4 RNA Ligase 1 components in combination with pCp-AF488. Reaction conditions: 50 pmol RNA oligo, 1000 pmol pCp-AF488, 1 mM ATP, 40 U T4 RNA Ligase 1, 1x T4 RNA Ligase 1 Reaction Buffer, 1.33 U RNAse inhibitor, 10 % DMSO, 15 % PEG8000. 18 h incubation at 16 °C. AF488-labeled RNA oligo was subsequently purified from crude reaction mix with Oligo Clean & Concentrator Columns (#4060, Zymo) and analyzed by UV/VIS spectroscopy (A260 nm: AF488-labeled & non-labeled RNA population/ A494 nm: AF488-labeled RNA probe only).

Table 1: Overview on available AF (Alexa Fluor®)-Dye labeled pCps.

Product Dye Substitute for Excmax/ Emmax
pCp-AF488 AF488
(also known as Alexa Fluor®488)
Fluorescein / ATTO488 494 / 517
pCp-AF555 AF555
(structural analog to Alexa Fluor®555)
Cy3 555 / 572
pCp-AF594 AF594
(also known as Alexa Fluor®594)
Texas Red 590 / 617
pCp-AF647 AF647
(structural analog to Alexa Fluor®647)
Cy5 / ATTO643 648 / 671
Cy®-Dye or ATTO®-Dye labeled pCps are available as well!

Questions or inquiries?

Dr. Barbara Zschoernig

Please contact Barbara with all questions or inquiries you may have!